Abstract

Inflammatory pain, such as arthritis pain, is a growing health problem1. Inflammatory pain is generally treated with opioids and cyclooxygenase (COX) inhibitors, but both are limited by side effects. Recently, resolvins, a novel family of lipid mediators including RvE1 and RvD1 derived from omega-3 polyunsaturated fatty acid, show remarkable potency in treating disease conditions associated with inflammation2, 3. Here we report that peripheral (intraplantar) or spinal (intrathecal) administration of RvE1 or RvD1 (0.3–20 ng) potently reduces inflammatory pain behaviors in mice induced by intraplantar injection of formalin, carrageenan or complete Freund’s adjuvant, without affecting basal pain perception. Intrathecal RvE1 also inhibits spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and TNF-α. RvE1 plays anti-inflammatory roles via reducing neutrophil infiltration, paw edema, and proinflammatory cytokine expression. RvE1 also abolishes TRPV1- and TNF-α-induced excitatory postsynaptic current increase and TNF-α-evoked NMDA receptor hyperactivity in spinal dorsal horn neurons, via inhibition of ERK signaling pathway. Thus, we demonstrate a novel role of resolvins in normalizing spinal synaptic plasticity that has been implicated in generating pain hypersensitivity. Given the remarkable potency of resolvins and well known side effects of opioids and COX inhibitors, resolvins may represent novel analgesics for treating inflammatory pain.

Resolution of acute inflammation, once thought to be a passive process, is now shown to involve active biochemical programs that enable inflamed tissues to return to homeostasis 2. The actions of pro-resolution mediators are in sharp contrast to those of currently used anti-inflammatory therapeutics. For example, inhibitors of COX and lipoxygenases disrupt resolution, because these enzymes are also required for the biosynthesis of pro-resolution mediators4–6. Resolvins, such as RvD1 and RvE1, are biosynthesized from omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), respectively, and show remarkable potency in resolving inflammation-related diseases such as periodontal diseases, asthma, and retinopathy 2, 3, 7. Peripheral and central mechanisms of inflammatory pain are not fully understood 8–11. Here, we examined whether peripheral and central resolvins can attenuate inflammatory pain, and further investigated how resolvins regulate synaptic plasticity in spinal cord dorsal horn neurons that has been strongly implicated in the generation of persistent pain 10, 11.

First, we examined the actions of RvE1 in an acute inflammatory pain condition induced by intraplantar injection of formalin. Formalin induced characteristic two-phase spontaneous pain behavior, and the second phase is likely mediated by spinal cord mechanisms12, 13. We delivered synthetic resolvins to the mouse spinal cord via intrathecal (i.t.) route using lumbar puncture14, 15. Preemptive injection of RvE1 at very low doses, only 0.3 and 1.0 ng (i.e. 1 and 3 pmol), reduced the 2nd but not the 1st phase pain behavior, suggesting a possible central action of RvE1 (Fig. 1a, b). Notably, the effective dose range of RvE1 was much lower than that of either morphine or the COX-2 inhibitor NS-398 (Fig. 1c).

In summation, these results demonstrated that resolvins, at very low doses (0.3–20 ng), effectively reduced inflammatory pain symptoms in several mouse models, via both peripheral and central actions. Biosynthesized during resolution of acute inflammation, resolvins are known to act on immune cells to produce anti-inflammatory actions (e.g., reducing polymorphonuclear leukocyte infiltration and tissue injury) and pro-resolving actions (e.g., increasing phagocytosis activity of macrophages)2. As expected, peripheral administration of RvE1 reduced CRG-elicited expression of proinflammatory cytokines, neutrophil infiltraton, and paw edema. Since the proinflammatory cytokines such as TNF-α and IL-1β are indispensable for the pathogenesis of inflammatory pain (Fig. 3a, b) 24, 31, resolvin’s antinociceptive actions could be attributable to its anti-inflammatory role. Hence it is particularly noteworthy that we demonstrated herein a novel mechanism in pain resolution in which RvE1 rapidly, within minutes, reduced inflammatory pain via modulating synaptic plasticity in dorsal horn neurons (Fig. 3h, ​,4f).4f). RvE1 not only abolished TRPV1-induced EPSC frequency increase and spontaneous pain, but also blocked TNF-α-induced EPSC frequency increase and NMDAR hyperactivity. As illustrated in Fig. 3h and Fig. 4f, RvE1 requires the activation of the GPCR ChemR23 and the inactivation of the ERK signaling pathway in both presynaptic and postsynaptic neurons for mediating its antinociceptive actions.

Current treatments for inflammatory pain are limited by side effects, such as respiratory depression, sedation, nausea, vomiting, constipation, dependence, tolerance, and addiction after opioid treatment 32, 33 and serious cardiovascular effects associated with long-term treatment of COX-2 inhibitors 1, 34. Also, COX-2 inhibitors and local anesthetic impair the resolution of acute inflammation4, 6. Although enthusiasm for TRPV1 antagonists is high, these drugs can cause hyperthermia 35 and have limited efficacy on mechanical allodynia. The present results show that resolvins are potent in attenuating inflammatory pain without changing basal pain sensitivity. Given the remarkable anti-hyperalgesic efficacy of resolvins and safety associated with endogenous mediators, resolvins and their metabolically stable analogues may represent a novel family of analgesics useful for treating inflammation-associated pain such as arthritic pain and postoperative pain. This new analgesic function gives a unique feature that now adds to the beneficial anti-inflammatory and pro-resolving actions of resolvins2.

Supplementary Material

Supplementary Data

Acknowledgments

The work was supported in part by US National Institutes of Health grants NIH R01-DE17794, R01-NS54362 to RRJ, and R37 GM38765, R01-DE019938, and R01-DK074448 to CNS, and NS67686 to both RRJ and CNS.

Footnotes

AUTHOR CONTRIBUTIONS

R.-R.J. and C.N.S. formulated the hypotheses, designed and supervised the project, and prepared the manuscript; R.-R.J. designed most experiments; Z.-Z.X., T.L. and J.Y.P. conducted behavioral studies; L.Z. performed electrophysiological studies; Z.-Z.X. and L.Z. performed immunohistochemistry; Z.-Z.X. performed siRNA knockdown, cytokine array, and Western blotting; T.B. performed in situ hybridization; R.Y. prepared resolvins and their analogs.

COMPETING INTERESTS STATEMENT

Resolvins are biotemplates for stable analogs. Patents on these are awarded and assigned to the Brigham and Women’s Hospital, and C.N.S. is the inventor. These patents are licensed for clinical development. Other authors declare that they have no competing financial interests.

12. Yamamoto T, Yaksh TL. Comparison of the antinociceptive effects of pre- and posttreatment with intrathecal morphine and MK801, an NMDA antagonist, on the formalin test in the rat. Anesthesiology. 1992;77:757–763.[PubMed]